- Short report
- Open Access
Extended blood circulation and joint accumulation of a p(HPMA-co-AzMA)-based nanoconjugate in a murine model of rheumatoid arthritis
© Ebbesen et al.; licensee BioMed Central Ltd. 2014
- Received: 24 June 2014
- Accepted: 3 September 2014
- Published: 11 September 2014
We recently synthesized a hydrophilic polymer, poly(N-(2-hydroxypropyl)methacrylamide-co-N-(3-azidopropyl)methacrylamide), p(HPMA-co-AzMA), by RAFT polymerization using a novel azide-containing methacrylamide monomer that through a post modification strategy using click chemistry enabled facile preparation of a panel of versatile and well-defined bioconjugates. In this work we screen a panel of different molecular weight (Mw) fluorescently tagged p(HPMA-co-AzMA) in healthy mice, by live bioimaging, to select an extended circulatory half-life material for investigating joint accumulation in a murine collagen antibody-induced arthritis model.
Fluorescence image analysis revealed half-lifes of <20 min, 2.8 h and 6.4 h for p(HPMA-co-AzMA) of 15, 36 and 54 kDa, respectively, with ~10% polymer retained in the blood after 24 h for the highest Mw. p(HPMA-co-AzMA) of 54 kDa showed enhanced accumulation in the joints of the arthritic mouse model with a bioavailability (AUC = 1783% · h) ~12 times higher (P = 0.01) than healthy control (AUC = 148% · h).
p(HPMA-co-AzMA) of 54 kDa exhibited extended circulatory half-life and preferential accumulation in inflamed joints of a murine model of rheumatoid arthritis (RA). This combined with well-defined polymer size and versatility for conjugation of a range of biomolecules promotes p(HPMA-co-AzMA) for potential applications in the delivery of drugs for treatment of RA.
- Collagen Antibody-Induced Arthritis
- Extended circulation
- in vivo
- Image analysis
- Joint accumulation
Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease affecting ~ 1% of the population causing cartilage and bone destruction in synovial joints . The pathogenesis involves synovial infiltration by circulatory immune cells that induce inflammation, modulated predominantly by proinflammatory cytokines such as tumor necrosis factor alfa (TNFα) [2, 3]. Common clinical treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying antirheumatic drugs (DMARDs) such as anti-TNFα antibodies. Although effective in many patients, these therapeutics have associated side-effects such as myelosuppression and increased infection risk, primarily due to a general systemic immune suppression that necessitates alternative approaches [1, 4]. RA associated angiogenesis, necessary for formation of pannus and invasion of inflammatory cells into the synovial tissue, has been identified to establish a macromolecular retention effect [5–7], conceptually similar to the EPR effect for tumors . Hydrophilic polymers synthesized with high molecular weights (Mw) enable the construction of polymer macromolecular drugs  with extended blood circulation and specific targeting to arthritic joints. This has been used for local suppression of proinflammatory cytokines such as TNFα  that may increase the clinical efficacy and reduce generalized side effects.
We hypothesize that the p(HPMA-co-AzMA) constructs will exhibit prolonged blood circulation and specific accumulation in inflamed joints in a murine model of rheumatoid arthritis supporting its potential therapeutic application for RA.
For blood circulation studies, a PBS solution (100 μL) of p(HPMA-co-AzMA) polymers (4 mg/kg) of different Mw (15, 36 and 54 kDa) labeled with an Atto680 dye (ATTO-TEC GmbH, Siegen, Germany), or PBS only control was administered intravenously (i.v.) in healthy BALB/c male mice (8 week-old, Taconic Europe, Ry, Denmark). Blood volumes (~80 μL, max 2 pr. animal) were sampled from 5 min up to 24 h post-injection and the blood plasma was subsequently transferred to narrow 20 uL glass capillary tubes (minicaps, Hirschmann) and imaged with the IVIS® Spectrum using a 675/720 nm filterset and analyzed using the Living Image software version 4.3 (PerkinElmer).
Nine week-old, male, DBA/1 (Taconic Europe, Ry, Denmark) were used for the collagen antibody-induced arthritis (CAIA) model  with intraperitoneal (i.p.) injection of the monoclonal antibody mix (Arthrogen-CIA® arthritogenic monoclonal antibody 5 clones cocktail kit, Chondrex, Inc. Redmond, USA) and a subsequent LPS injection 3 days later. The severity of the arthritic condition was monitored by daily clinical scoring of joints (1–4)  with symptoms developing ~ day 3 and peaking around day 8. Healthy control mice (DBA/1) were injected i.p. with PBS.
For the joint accumulation studies, a PBS solution of the 54 kDa Atto680-labeled (0.57 w/w%) p(HPMA-co-AzMA), or the free dye, was administered by intravenous (i.v.) injection to arthritic and control animals at a polymer dose of 4 mg/kg or the dye-equivalent amount (22.6 ug/kg) of free dye. This dose was comparable to existing studies [17, 18] and at a concentration by which the polymer conjugated dye could be easily visualized using in vivo fluorescence imaging. Joint fluorescence was monitored before, and up to, 24 h post-injection by IVIS Spectrum in vivo imaging of isoflurane (2.5%) anesthetized mice. Multispectral fluorescence images covering the absorption-emission profile of the Atto680 dye were acquired using the Living Image software (PerkinElmer). Images were spectrally unmixed to subtract background fluorescence, which enabled the total fluorescence emission from joint areas from each mouse to be quantified. Measured intensities of the fluorescence emission are generally linear dependent on fluorophore concentration, extinction coefficient and quantum yield, which enables a direct correlation to polymer levels in the blood or tissue .
All image data was analyzed using Prism (GraphPad Software Inc.). Blood polymer levels were normalized to the value measured at 5 min and joint polymer levels to the highest mean value and presented as % with SEM. Students t-test was performed to determine data significance.
All procedures of animal work were performed according to international recognized guidelines and the animal experimental protocols approved by ‘The Experimental Animal Inspectorate in Denmark’ under The Danish Veterinary and Food Administration, Ministry of Food, Agriculture and Fisheries (Registration number: 2013 - 15 - 2934 - 00789-C2, issue date: March 5, 2013).
Results and discussion
In summary, this work demonstrates the in vivo characteristics of a new azide containing copolymer, (p(HPMA-co-AzMA)), which offers a more versatile and well-defined alternative to existing bioconjugate systems. Attractive in vivo properties such as prolonged blood retention and specific accumulation in inflamed joints of RA illustrate the potential of the material for systemic delivery of anti-inflammatory drugs for local effects in the treatment of RA.
We thank the Lundbeck Foundation for supporting this work through the grant: Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration.
- O’Dell JR: Therapeutic Strategies for Rheumatoid Arthritis. N Engl J Med. 2004, 350: 2591-2602. 10.1056/NEJMra040226.View ArticlePubMedGoogle Scholar
- Arend WP: Physiology of cytokine pathways in rheumatoid arthritis. Arthritis Rheum-Arthritis Care Res. 2001, 45: 101-106. 10.1002/1529-0131(200102)45:1<101::AID-ANR90>3.0.CO;2-7.View ArticleGoogle Scholar
- Howard KA, Paludan SR, Behlke MA, Besenbacher F, Deleuran B, Kjems J: Chitosan/siRNA nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model. Mol Ther. 2009, 17: 162-168. 10.1038/mt.2008.220.PubMed CentralView ArticlePubMedGoogle Scholar
- Olsen NJ, Stein CM: New drugs for rheumatoid arthritis. N Engl J Med. 2004, 350: 2167-2179. 10.1056/NEJMra032906.View ArticlePubMedGoogle Scholar
- Wang D, Miller SC, Sima M, Parker D, Buswell H, Goodrich KC, Kope P: The arthrotropism of macromolecules in adjuvant-induced arthritis rat model: a preliminary study. Pharm Res. 2004, 21: 1741-1749.View ArticlePubMedGoogle Scholar
- Paleolog EM: Angiogenesis in rheumatoid arthritis. Arthritis Res. 2002, 4: S81-S90. 10.1186/ar575.PubMed CentralView ArticlePubMedGoogle Scholar
- Koch AE: Angiogenesis as a target in rheumatoid arthritis. Ann Rheum Dis. 2003, 62: ii60-ii67.PubMed CentralView ArticlePubMedGoogle Scholar
- Fang J, Nakamura H, Maeda H: The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv Drug Deliv Rev. 2011, 63: 136-151. 10.1016/j.addr.2010.04.009.View ArticlePubMedGoogle Scholar
- Yuan F, Quan L-d, Cui L, Goldring SR, Wang D: Development of macromolecular prodrug for rheumatoid arthritis. Adv Drug Deliv Rev. 2012, 64: 1205-1219. 10.1016/j.addr.2012.03.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Choy EHS, Hazleman B, Smith M, Moss K, Lisi L, Scott DGI, Patel J, Sopwith M, Isenberg DA: Efficacy of a novel PEGylated humanized anti‒TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II double‒blinded, randomized, dose‒escalating trial. Rheumatology. 2002, 41: 1133-1137. 10.1093/rheumatology/41.10.1133.View ArticlePubMedGoogle Scholar
- Duncan R, Vicent MJ: Polymer therapeutics-prospects for 21st century: the end of the beginning. Adv Drug Deliv Rev. 2013, 65: 60-70. 10.1016/j.addr.2012.08.012.View ArticlePubMedGoogle Scholar
- Barz M, Luxenhofer R, Zentel R, Vicent MJ: Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure–property relationships to better defined therapeutics. Polym Chem. 2011, 2: 1900-1918. 10.1039/c0py00406e.View ArticleGoogle Scholar
- Ebbesen MF, Schaffert DH, Crowley ML, Oupický D, Howard KA: Synthesis of click-reactive HPMA copolymers using RAFT polymerization for drug delivery applications. J Polym Sci A Polym Chem. 2013, 51: 5091-5099. 10.1002/pola.26941.View ArticleGoogle Scholar
- Wang D, Miller S, Liu X-M, Anderson B, Wang XS, Goldring S: Novel dexamethasone-HPMA copolymer conjugate and its potential application in treatment of rheumatoid arthritis. Arthritis Res Ther. 2007, 9: R2-10.1186/ar2106.PubMed CentralView ArticlePubMedGoogle Scholar
- L-d Q, Yuan F, Liu X-m, Huang J-g, Alnouti Y, Wang D: Pharmacokinetic and biodistribution studies of N-(2-hydroxypropyl)methacrylamide copolymer-Dexamethasone conjugates in adjuvant-induced arthritis Rat model. Mol Pharm. 2010, 7: 1041-1049. 10.1021/mp100132h.View ArticleGoogle Scholar
- Khachigian LM: Collagen antibody-induced arthritis. Nat Protoc. 2006, 1: 2512-2516. 10.1038/nprot.2006.393.View ArticlePubMedGoogle Scholar
- Noguchi Y, Wu J, Duncan R, Strohalm J, Ulbrich K, Akaike T, Maeda H: Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues. Jpn J Cancer Res. 1998, 89: 307-314. 10.1111/j.1349-7006.1998.tb00563.x.View ArticlePubMedGoogle Scholar
- Shiah JG, Dvořák M, Kopečková P, Sun Y, Peterson CM, Kopeček J: Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer–doxorubicin conjugates in nude mice. Eur J Cancer. 2001, 37: 131-139. 10.1016/S0959-8049(00)00374-9.View ArticlePubMedGoogle Scholar
- Leblond F, Davis SC, Valdés PA, Pogue BW: Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications. J Photochem Photobiol B Biol. 2010, 98: 77-94. 10.1016/j.jphotobiol.2009.11.007.View ArticleGoogle Scholar
- Etrych T, Šubr V, Strohalm J, Šírová M, Říhová B, Ulbrich K: HPMA copolymer-doxorubicin conjugates: the effects of molecular weight and architecture on biodistribution and in vivo activity. J Control Release. 2012, 164: 346-354. 10.1016/j.jconrel.2012.06.029.View ArticlePubMedGoogle Scholar
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